Further Reading
Nearly all the cell-types of the diffuse endocrine system (DES) are
argyrophilic (9, 10), as are the axons of neurons and reticulin, a term
the first, which is often called impregnation, the tissue (whole pieces
in the UK.) These complex silver ions are more easily reduced than
(Puchtler et al 1986).
atoms of the metal and far too small to be visible. In the second step,
How does H & E work? Again starting with the easy bit: eosin
stains sections by acid dyeing. The Al-hematein staining mechanism
nuclear and Nissl staining basic dyes such as cresyl violet or toluidine
the adrenal cortex and the embryonic eye. Tiny fragments of fresh
such basic dyes, but does not eliminate Al-hematein staining. Routine
by the action of light, discoloring all parts of the tissue. This slower
The same holds for von Kossas stain for calcified material. In this,
| 167
References
6. Lundqvist M, Arnberg H, Candell J, et al. Silver stains for identification of neuroendocrine cells. A study of the chemical background.
Histochem J 1990;38:615-623.
The reaction with sulfurous acid (Fig. 1, V) decolorizes the dye. The
structure of the colorless product was debated for many years (4),
Figure 1. The components of basic fuchsine (I - IV) and the formation of Schiffs reagent by reaction of pararosaniline with sulfurous acid (V,VI).
| 169
be stable while exposed to the air and losing sulfur dioxide for 15
is the zwitterionic form shown (VI) in which one of the amino groups
is protonated (6).
stain must be acidic (pH about 2 is best for most applications) and it
matrix, mast cell granules and other materials that are stainable by
formerly commonplace (10, 11) but they weaken the final color. Rapid
must contain more sulfurous acid than was needed to decolorize the
so Schiffs reagent that has become pink from losing SO2 can be
after a few months of storage (4, 9). In equation VII it is seen that a
the color. The same effect can be achieved in 15 seconds with 0.3%
original dye. Some of the equilibria that account for this instability
towards neutrality. SO2 can escape from a loosely capped bottle. The
purposes high sensitivity is not usually needed and the reagent must
Schiffs reagent then becomes pink and is unfit for use because it
SOCl2, to the dye solution. This liquid reacts instantly with water:
4. Kasten, FH. The Chemistry of Schiffs reagent. Int Rev Cytol 1960;10:1-100.
with thionyl chloride can be stored for a few years in securely capped
bottles (4, 9). Probably the most popular recipes for Schiffs reagent
(4, 7, 10) are those in which the sulfurous acid is formed by dissolving sodium metabisulfite in the dye solution and then adding
hydrochloric acid, allowing control of both the pH and sulfurous
acid content as well as avoiding the use of more hazardous
chemicals. Many vendors sell Schiffs reagent as a ready-made
solution. A survey of 20 material safety data sheets (MSDS) shows
that all were made with sodium metabisulfite, bisulfite or sulfite
(0.1-2.0%), and hydrochloric acid, with concentrations of basic
fuchsine ranging from 0.01 to 1.0%. The pH was reported in only
6. Robins JH, Abrams GD, Pinock JA. The structure of Schiff reagent
aldehyde adducts and the mechanism of the Schiff reaction as
determined by nuclear magnetic resonance spectroscopy. Can J Chem
1980;58:339-47.
7. Pearse AGE, Stoward PJ. Histochemistry, Theoretical and Applied, 4th
edn. Vol. 2. Analytical Technique. Edinburgh: Churchill-Livingstone,
1985 pp. 850-852.
8. Heinemann RL. Comparative spectroscopy of basic fuchsin, Schiff
reagent, and a formalin-Schiff derivative in relation to dye structure and
staining. Stain Technol 1970;45(4):165-72.
9. Kiernan JA. Histological and Histochemical Methods: Theory and
Practice. 4th ed. Bloxham, UK: Scion, 2008, pp. 224-227, 293-296.
were reported for the Feulgen technique (4). Current practice favors
reagent (VI, VIII, IX) and on the product of reaction with tissue-bound aldehyde groups (X, XI).
References
| 171
Giemsas Formulation
Gustav Giemsa (1867-1948) endeavored to produce mixtures of dyes
that would reliably provide the Romanowsky color scheme (6). These
formulations were published in 1902-1904, with the most advanced
version being an aqueous solution containing a large excess of
azure II over eosin Y. Azure II was a deliberate mixture of unoxidized
methylene blue with azure I. Azure I was the name used at that time
for the dye now called azure B. Modern formulations of Giemsas
stain are made by mixing azure B eosinate with methylene blue and
are commercially available as Giemsa powder, which is dissolved in
solution old enough to have mold growing on its surface. The improved
stock solution (9). For staining, the stock solution is diluted in water that
by Unna (3), who also published in 1891 and found that the aging
blue and eosin are unstable because the oppositely charged dye ions
Pure azure B is more expensive than the crude variety, and there
blue. Later work showed that all commercially produced thiazine dyes
related blood stains made from polychrome methylene blue and from
Figure 1. Dyes present in Giemsas and other blood stains. The arrows indicate the progressive demethylation and oxidation that constitute polychroming of
methylene blue.
| 173
among different suppliers and even among different lots from the
for Giemsas Jenners and Wrights stains are tested by the Biological
DNA in the nuclei of protozoa (18, 19) and some invertebrates (20).
Staining Mechanisms
Studies of the interaction of DNA with azure B (14, 15) indicate that
in solution the dye cations are present as dimers, each with the two
and cartilage. The colors are not the same as those seen in alcohol-
planar ring systems held together by van der Waals forces. The dimers
with the purine and pyrimidine rings of the DNA bases. Attraction
differ from their animal counterparts in not being associated with basic
B dimers changes the color of the stained DNA from blue to purple
bacteria probably also bind cationic dyes, and the phosphate groups
and guanidino groups (mainly side chains of lysine and arginine) over
negative organisms.
Figure 3. Karyogram of GTG-banded human chromosomes ( ) (46 chromosomes, including XY): GTG = G-banding with trypsin-Giemsa. Courtesy of Dr. Stefan Mueller,
Ludwig-Maximilians-University, Munich, Germany.
| 175
Chromosome Banding
Giemsa (G-banding) is the stain most commonly used to display
transverse bands in mitotic (metaphase) chromosomes, having
largely replaced quinacrine and other fluorochromes (Q-banding)
that were formerly used for this purpose. There are various empirically
developed G-banding techniques in which chromosome preparations
typically cell cultures that have been arrested in metaphase, spread
onto slides, and fixed in 3:1 methanol-acetic acid are subjected to
pre-treatments before staining with Giemsa (25, 26). The dye cations
in the Giemsa mixture (azure B and methylene blue) are attracted by
the phosphate anions of DNA and are then held more closely by van
der Waals forces to regions rich in adenine and thymine. Regions of
a chromosome rich in guanine-cytosine pairs do not bind the blue
dyes. The pretreatment determines the type of banding pattern. In
typical G-banding the preparations are treated either with a sodium
chloride and citrate solution at 60C for one hour or with trypsin for 10
seconds at room temperature before staining in dilute Giemsa at pH
6.8 for 45 minutes (Fig. 3). R-banding is a method that shows bands
that are complementary to the G-bands; that is, the dark and light
regions of the G-banded chromosome are reversed. The pretreatment
for R-banding is with 1.0 M NaH2PO4 (pH about 4) at 88C for 20
minutes, and the staining is with a more concentrated Giemsa
solution, for 10 minutes (27). G-banding is the method usually used for
identifying chromosomes. R-banding is useful for showing the ends of
chromosomes (telomeres). A variant known as T-banding emphasizes
these terminal segments; after treatment with a phosphate buffer,
pH 5.1 at 87C for 20-60 minutes the preparations are stained in
Giemsa, also at 87C (25). The histochemical rationales of the various
pretreatments have not been systematically investigated (26).
10. Green FJ. The Sigma-Aldrich Handbook of Stains, Dyes and Indicators.
Milwaukee, Wisconsin: Aldrich Chemical Company, 1990: 109-122,
388-389, 413-414, 448-450, 734-736.
that in water may interfere with dye uptake, and 3) they tend to stain
in dilute acid to pull out the excess color from the chromatin and
12. Wittekind DH, Kretschmer V. On the nature of RomanowskyGiemsa staining and the Romanowsky-Giemsa effect. II. A revised
Romanowsky-Giemsa staining procedure. Histochem J 1987;19:399-401.
13. Penney DP, Powers JM, Frank M, Churukian C. Analysis and testing
of biological stains the Biological Stain Commission procedures.
Biotech Histochem 2002;77(5-6):237-275.
References
1. Woronzoff-Dashkoff KPK. The Ehrlich-Chenzinzky-Plehn-MalachowskiRomanowsky-Nocht-Jenner-May-Grunwald-Leishman-Reuter-WrightGiemsa-Lillie-Roe-Wilcox stain. The mystery unfolds. Clin Lab Med
1993;13(4):759-771.
2. Wittekind DH. On the nature of Romanowsky-Giemsa staining and its
significance for cytochemistry and histochemistry: an overall review.
Histochem J 1983;15:1029-1047.
3. Unna PC. Uber die Reifung unsrer Farbstoffe. Z wiss Mikr 1891;8:475-487.
25. Gosden JR, ed. Chromosome Analysis Protocols. Totoya, NJ: Humana
Press, 1994: 59-81.
6. Barcia JJ. The Giemsa stain: its history and applications. Int J Surg Path
2007;15(3):292-296.
that can be applied for many minutes without overstaining and without
of stain, 2) select suitable staining times, 3) find out how many slides
not necessary for the remainder of the life of the particular stain that
Simply looking at one of the first slides stained daily and initialing a
stain quality log sheet is of no value if a laboratory has not defined its
See Table 2.
| 177